JPH04305528A - Proliferation stimulant for keratinocyte and dermal fibroblast - Google Patents

Abstract

PURPOSE:To obtain a proliferation stimulant for keratinocytes and dermal fibroblasts, containing a specific adenosine-3',5'-cyclic phosphate (AMP) derivative as an active ingredient and effective against incised wounds caused by surgical operation, burns, traumas caused by injuries, cold injuries, mucosal diseases, hemorrhoids, etc. CONSTITUTION:A proliferation stimulant for keratinocytes and dermal fibroblasts containing an adenosine-3',5'-cyclic phosphate derivative (e.g. N<6>, 2-0-dibutyryl cAMP in an amount of preferably about 0.0001-1wt.%) expressed by the formula (R<1> is H, 1-6C alkyl or 2-7C aliphatic acyl; R<2> is H, mercapto, 1-6C alkylthio, benzylthio, amino, OH, Cl or Br; R<3> is H or 2-7C aliphatic acyl; X is H or alkali metal, provided that R<1> to R<3> will not simultaneously be H) as an active ingredient. The aforementioned medicine is preferably used in the form of external use. An epidermal growth factor or a growth factor derived from blood platelets is preferably further blended in the aforementioned adenosine-3',5'-cyclic phosphate derivative expressed by the formula.

Description

[Detailed description of the invention]

0001

[Industrial Application Field] The present invention relates to a keratinocyte and dermal fibroblast proliferation stimulant, and more particularly to a keratinocyte and dermal fibroblast proliferation stimulator that stimulates and promotes the proliferation of keratinocytes and dermal fibroblasts in various skin injured conditions. In particular, it relates to agents for promoting the healing of skin injuries.

0002

BACKGROUND OF THE INVENTION There are a variety of local skin conditions and injuries that require treatment. For example, surgical wounds, burns, trauma wounds, cold injuries, mucosal diseases, hemorrhoids, etc. are examples of local skin conditions or injuries that require treatment.

0003

[Problems to be Solved by the Invention] There are no fully satisfactory treatments for these skin conditions, and it has been desired to develop treatment means using a new approach.

0004

[Means for Solving the Problems] The present inventors have conducted various studies on the regeneration function of skin tissue at the cellular level, and have found that the proliferation of keratinocytes and fibroblasts is deeply involved in the healing process of skin tissue wounds. They found that stimulating the proliferation of these cells significantly promotes wound healing. Furthermore, the following general formula (1
The present inventors have discovered that the adenosine-3',5'-cyclic phosphoric acid derivative of ) has the effect of stimulating the proliferation of these cells and is useful for promoting healing of skin injuries, leading to the completion of the present invention.

That is, the present invention is based on the following general formula (1)

0
006]

[Chemical 2]

[In the formula, R1 is a hydrogen atom, and has 1 to 6 carbon atoms]
represents an alkyl group or an aliphatic acyl group having 2 to 7 carbon atoms, R2 represents a hydrogen atom, a mercapto group, an alkylthio group having 1 to 6 carbon atoms, a benzylthio group, an amino group, a hydroxyl group, a chlorine atom or a bromine atom, R3 represents a hydrogen atom or an aliphatic acyl group having 2 to 7 carbon atoms, and X represents a hydrogen atom or an alkali metal atom. However, R1, R2 and R3
cannot become hydrogen atoms at the same time. ] is an adenosine-3',5'-cyclic phosphate derivative (hereinafter referred to as cA
The present invention provides a keratinocyte and skin fibroblast growth stimulant containing a MP derivative (abbreviated as MP derivative) as an active ingredient.

cAMP derivative (1) used in the present invention
is a known compound, for example, Japanese Patent Publication No. 50-2255
Publication No. 9, Japan Clinical Volume 40, No. 11, pp. 14-19 (
1982), Journal of Cycli
c Nucleotide Research, 2
, 307-319 (1976), Biochem. Bi
ophys. Acta. , 148, 99-105 (
1967) and others. Among these cAMP derivatives (1), a compound in which R1 and R3 are n-butyryl groups, R2 is a hydrogen atom, and X is a sodium atom (hereinafter sometimes abbreviated as dbcAMP) is particularly preferred.

As shown in the Examples below, cAMP derivative (1) promotes the proliferation of mammalian keratinocytes and skin fibroblasts. Furthermore, such cell division-promoting effects are caused by epidermal growth factor (EGF) and platelet-derived growth factor (P).
DGF). Surprisingly, these derivatives also contain biologically active TGF-
It was also found to increase levels of beta, a peptide that significantly stimulates the formation of intercellular stroma.

[0010] Based on these remarkable findings, it is believed that cAMP derivative (1) is useful for healing the various wounds described above for several reasons as described below. (1) Keratinocytes appear to play a major role in the final stage of healing, or re-epithelialization, of mammalian skin. The effect of keratinocyte proliferation therefore promotes re-epithelialization. (2) Since keratinocytes migrate along the surface of granular tissue, granular tissue is first required, and skin fibroblasts play an important role in the formation of granular tissue. Dermal fibroblasts secrete collagen and glycosaminoglycans, which are the main connective substances (interstitium) that support granular tissue. (3) Various endogenous growth factors such as EGF and PDGF are thought to be involved in the wound healing process. cAM
The cell division promoting effect of P derivative (1) is similar to that of EGF and PDGF.
increases in the presence of EGF stimulates keratinocyte proliferation. PDGF is believed to have numerous biological activities. That is, it not only stimulates cell division, but also changes the intercellular interstitium of tissues, and acts as a chemotactic factor for neutrophils, monocytes, smooth muscle cells, and fibroblasts. (4) TGF-beta stimulates fibroblast proliferation. Additionally, TGF-beta promotes the production and inhibits the degradation of glycosaminoglycans, collagen, and fibronectin. Therefore, TGF-
Increased beta increases the connective tissue (which is responsible for normal wound healing).
Contributes to the formation of intercellular stroma).

The cAMP derivative (1) of the present invention has the effect of stimulating the proliferation of keratinocytes and skin fibroblasts in mammals in need of treatment. Because it stimulates the proliferation of keratinocytes and skin fibroblasts, this cAMP derivative (1) can be used in surgical incisions,
It can be used to treat skin injuries such as burns, injury wounds, cold injuries, mucosal diseases, and hemorrhoids. For example, the fact that cAMP derivative (1) showed a beneficial effect on the tensile strength of the healed area of incisional wounds in experimental animals means that cAMP derivative (1) also has a beneficial effect on the tensile strength of the healed area of incisional wounds in humans. It has been shown to heal wounds. Furthermore, in animals such as pigs, cAMP derivative (1) has shown a good effect on the healing rate of skin wounds. It is clear that it promotes the healing of burns, trauma wounds, cold injuries, mucosal diseases, and hemorrhoids.

[0012] Here, the term "surgical wound" mainly refers to, for example,
This includes wounds caused by appendectomy, cholecystectomy, episiotomy, hysterectomy, etc., and wounds at skin graft sites and skin donor sites. It also includes other surgical wounds. "Wounds caused by injury" mainly include all wounds caused by damage to the skin caused by metal, wood, glass, plastic, etc. "Mucosal disease" is
Examples include aphthous ulcers and herpetic ulcers. Furthermore, in the present invention, hemorrhoids include both external and internal hemorrhoids.

As described above, the therapeutic agent of the present invention is particularly advantageous when applied to humans, but the cAMP derivative (1) can be used to promote the proliferation of keratinocytes and skin fibroblasts in any mammal. You may. That is, the present invention can also be applied veterinarily to dogs, cats, horses, cows, pigs, etc.

In the present invention, the cAMP derivative (1) can be formulated into various external preparations such as solutions, emulsions, ointments, creams, lotions, and poultices. That is, one or more types of cAMP derivatives (1) may be mixed into an external base to form a formulation. The base is not particularly limited as long as it is a known external base. Particularly preferred formulations include one or more c.
This is a solution prepared by dissolving the AMP derivative (1) in physiological saline, or an ointment using macrogol as a base.

Generally, cAMP derivatives (1) to be prepared
The amount of can vary over a wide range, but is usually about 0.00001 to 10%, preferably about 0.0001 to 1% by weight of the total base weight. Generally, the therapeutic agent of the present invention is applied to the affected area once to several times a day. The amount of one application is 100cm2 depending on the degree of scratches.
It is preferable that cAMP derivative (1) is contained in an amount of 0.01 mg to 1 g, particularly 1 to 100 mg. c.
When the AMP derivative (1) is used in combination with neither EGF nor PDGF, it is generally preferred that the cAMP derivative (1) is present in an amount of about 0.0001 to 0.01% by weight based on the total base weight. When EGF or PDGF (preferably EGF) is used in combination, the amount of cAMP derivative (1) is about 0.00002 to 1 based on the total base weight.
It is preferable to use it in a state containing % by weight. According to the present invention, any known base may be used to prepare the cA of the present invention.
MP derivative (1) alone, and optionally EGF and PD
A formulation may be prepared by combining both or either one of the GFs. For example, formulations may be prepared according to US Pat. No. 4,126,702 and/or US Pat. No. 4,760,096. The formulation base may be in the form of a cream, lotion, ointment, solution, or even a soft solid bar.

[0016]

[Examples] Next, the present invention will be explained in detail by giving examples regarding pharmacological tests and preparations, but the present invention is not limited thereto.

Pharmacological test 1 (1) Method Fibroblast experiment Monolayer culture of adult skin fibroblasts was carried out using Falanga (Fa
Using the known method of Langa et al., explant culture of the skin and subcutaneous tissue of the back of the forearm, which were specimens for biopsy, was performed. J. Invest. Dermatol. ，
89, 100-104 (1987). The cultured fibroblasts used in this experiment were obtained from three donors and were not used beyond the 8th in vitro passage. Fibroblast proliferation was determined by cell count and 3H-thymidine incorporation on tissue culture plastic or collagen gels. In the number counting experiment, fetal bovine serum [FBS
It is abbreviated as Gibco Laboratories (
Dulbecco's Modified Eagle's Medium (abbreviated as DME) with 10% Dulbecco's Modified Eagle's Medium (DME, Grand Island, New York)
o Laboratories), 2.1 cm2
5000 fibroblasts were seeded per well. After 20 hours, the medium was mixed with DME alone (control) plus 3% FBS or dbcAMP at 10-6 to 10-3.
Replaced with one added within the range of M. In all experiments, some cells were incubated in DME without serum as another control. 4 for each experimental group
The number of cells in each well was counted every 1 to 2 days using a hemocytometer. Four days after plating the cells, fresh medium was added to those with and without dbcAMP. For experiments measuring 3H-thymidine incorporation, cells were plated on tissue culture plastic or collagen gels and incubated in DME with 10% FBS. After 20 hours, the medium was
In the same manner as above, DME was replaced with 3% FBS plus DME alone or dbcAMP plus DME. After overnight incubation, 3H-thymidine was added to the cultured cells. In experiments using growth factors, human epidermal growth factor [EG
Abbreviated as F. ICN Biomedicals (Costa Mesa, CA)] 5 ng/ml, human platelet-derived growth factor [abbreviated as PDGF]. ICN Biom
edicals] 4 ng/ml, or transforming growth factor-beta 1 [abbreviated as TGF-beta 1]. R&D S
systems (Minneapolis, Minnesota)〕5ng
/ml either alone or in combination with dbcAMP was added to cultured cells.

Keratinocyte experiment Cultured human keratinocytes of in vitro 2nd or 3rd passage derived from adult chest skin [Clonetics Corp.
San Diego, California) was used. The cells were grown in keratinocyte culture medium [abbreviated as KGM]. Cloneti
cs). KGM is EGF (.1ng/m
l), an optimal medium containing insulin (5.0 μg/ml), hydrocortisone (0.5 μg/ml), 0.15 mM calcium, and pituitary gland extract. For experiments measuring 3H-thymidine incorporation, 20,000 cells per 2.1 cm2 well were seeded in KGM. In experiments to determine cell counts, 5000 keratinocytes were seeded in KGM per 2.1 cm2 well. Similarly to the fibroblast counting experiment described above, after 20 hours, the culture medium was mixed with KGM alone (control) or dbcAMP at 1
It was replaced with KGM containing concentrations ranging from 0-6 to 10-3M. Four days after plating the cells, fresh medium with and without dbcAMP was added.

Quantitative analysis of 3H-thymidine In this experiment, fibroblasts and keratinocytes were
Twenty thousand cells per m2 well were plated in optimal medium and incubated overnight. The next day, the optimal medium was removed and replaced with treatment groups as above and incubated for 20 hours. The next day, the specific radioactivity was 87 Ci/mmol.
[Methyl-3H]thymidine [Amersham, (
Arlington Heights, IL] 1 μCi was applied to cultured cells for 3 hours. Wash cultured cells twice with PBS, 5%
Fixed twice with 1 ml of methanol solution of trichloroacetic acid,
Treated with 1 ml of 0.3N NaOH for 10 minutes at room temperature. Remove 0.8 ml from each well, add to 9 ml of Aquasol 2 (Dupont, Boston, MA), and add to scintillation counter 1211 Rackbeta (Pha).
rmaciaLKB, Inc. (Gatorsburg, MD)] with a coefficient efficiency of 65% for 3H. Results are expressed in dpm.

Collagen gel fibroblasts were cultured in collagen stroma prior to 3H-thymidine uptake measurements. Bovine skin collagen [Vitrogen 100, Collagen
Agen Corp., (Palo Alto, Calif.)] was used according to the manufacturer's guidelines. That is, 8 ml of Vitrogen 100 is mixed with 1 ml of 0.1
Mix in 1 ml of 10x phosphate buffered saline with MNaOH and pH 7 with 0.1 M NaOH or 0.1 M HCl.
．． Adjusted to 4. A pellet of cultured fibroblasts containing a predetermined number of cells was resuspended in Vitrogen 100 adjustment solution, and 2.1
0.25ml of 30,000 cells per cm2 well
It was sown in aliquots. This fibroblast-containing collagen suspension was allowed to gel at 37 degrees Celsius for 30 minutes. Additionally, 0.8 ml of DME containing 10% FBS was added to the medium. It was confirmed under a microscope that the cells were embedded in the collagen interstitium. After 20 hours, the wells were washed 8 times with DME alone for 2 hours. After that, 3%F
The cells were incubated with DME containing BS with or without dbcAMP. The experimental conditions for measuring 3H-thymidine incorporation are the same as in the above case.

[0021] TGF-beta measurement conditioned medium was prepared from 25 cm2 of confluent dermal fibroblast culture medium.
prepared in a flask. DME containing 10% FBS
A monolayer of fibroblasts cultured in was washed with serum-free DME every hour for a total of 6 times. After washing, it was confirmed that there were no abnormalities in cell morphology. And DME alone or 10-
Culture cells in 2 ml of DME containing 6MdbcAMP for 24 hours.
After treatment for an hour, the medium was collected and stored at 70 degrees Celsius. Cell numbers were determined using a hemocytometer. A published method [9] was used to measure TGF-beta. That is, Mv1Lu mink lung epithelial cells (CCL-64: Ame
ricanType Culture Colle
ction (Rocksville, Maryland)] 3H
- A TGF-beta inhibition assay was used that takes advantage of the strong inhibitory effect of TGF-beta on thymidine uptake. The lung epithelial cells were treated with DME, 0.2% FBS, 10mM
2 in assay medium consisting of HEPES (pH 7.4).
．． 50,000 cells were plated per 1 cm2 well. After being left in the presence of 5% CO2 at 37 degrees Celsius for 4 hours,
A decreasing concentration gradient of TGF-beta (0.0
A starting concentration of 150 pg/well in 8 ml) or 0.08 ml of 1:1 multiple dilutions of conditioned medium was added. Conditioned media were tested before and after acidification to activate latent TGF-beta. After 22 hours of incubation, 2
0.5 μCi of [methyl-3H]thymidine was added to each well over time. Then, methanol-acetic acid (3
:1v/v) of radioactive medium to directly add and fix the cells,
It was left at room temperature for 1 hour. After fixation, cells were diluted with 1.5 ml of 80
% methanol twice. Completely remove methanol and add 0.5 ml of trypsin (
0.2 mg/ml (GIBCO) was added to each well. After 1 hour incubation at room temperature, each well was
．． 5ml of 1% SDS was added. After 5 minutes, 0.8 ml of the solution was taken, added to 9 ml of Aquasol A, and counted using a scintillation counter (LKB).

Statistical analysis Results were evaluated using Student's t-test. The presence/absence test was based on p<0.05.

(2) Results The results obtained are shown in FIGS. 1 to 6. From FIG. 1, it can be seen from the number of cells that dbcAMP stimulated the proliferation of human keratinocytes compared to the case of KGM alone. This proliferative effect was dose-dependent, with maximum stimulatory effects observed at 10-5 and 10-6 M (p<0.02). Higher dbcAMP concentrations inhibited cell growth (p<0.001). 10-5M and 10-6M
The cell division-promoting effect of dbcAMP became statistically significant on day 3, ie, 2 days after adding dbcAMP to the medium, and continued until day 5 (p<0.05) and further to day 7. This growth-promoting activity of dbcAMP was also confirmed by measuring 3H-thymidine uptake in cultured subfusion keratinocytes. As shown in Figure 2, compared to KGM alone, dbcAMP caused an increase in 3H-thymidine uptake in a dose-dependent manner;
dbcAMP showed the greatest stimulatory effect (p<0.05). As in the cell number experiments, high concentrations of dbcAMP inhibit 3H-thymidine incorporation and 10-
3M showed the maximum inhibitory effect (p<0.001).

dbcAMP given to human skin fibroblasts
The effect is similar to that observed in human keratinocytes. As shown in Figure 3, 10-5 M and 10
-6M dbcAMP showed a stimulatory effect (p<0.00
1), 10-3M was inhibitory (p<0.001)
. In experiments examining cell numbers (data not shown),
dbcAMP at 10-6 M was stimulatory on fibroblast growth 3 days after seeding (p<0.05). However, no further effects appeared after that. Furthermore, even when cultured with collagen (Fig. 4), 10-6 M db
We confirmed that cAMP promoted cell proliferation (p<0.02) and the inhibition pattern at high concentrations was the same as when cultured on tissue culture plastic. The stimulatory effect of dbcAMP on fibroblasts was enhanced by adding a competing factor, platelet-derived growth factor (PDGF), or a promoting factor, epidermal growth factor (EGF) (Figure 5).
. In Figure 5, the control is 1% FBS. The stimulatory effect of PDGF is almost the same as the effect of combining 10-6MdbcAMP and PDGF. In contrast, EGF and 10
The combination with -5M or 10-6MdbcAMP is 3
Increased H-thymidine uptake. Separate experiments (not shown) using 3% FBS as a control did not show an increase in dbcAMP-induced cell proliferation by these growth factors. Interestingly, in the series of experiments shown in Figure 6, E
By adding GF or PDGF, 10-3M and 1
It was found that the inhibitory effect of 0-4M dbcAMP was partially or completely abolished. On the other hand, TGF-beta is
Increased growth inhibition with 10-3M and 10-4MdbcAMP. The concentration of dbcAMP, which normally has a stimulatory effect (
10-5M and 10-6M) could not overcome the inhibitory effect of TGF-beta. It is widely accepted that TGF-beta is a growth inhibitor in many cell types. Therefore, we investigated the release of TGF-beta from fibroblasts in the presence of dbcAMP and
It was confirmed whether the growth inhibiting or promoting effect of bcAMP was due to an increase or decrease in the amount of TGF-beta released. Expressed in picograms per 105 fibroblasts, total TGF-beta (latent and free) was 11% in controls.
50 pg, and 488 pg in the presence of dbcAMP. However, biologically active (free) TGF-beta was 80 pg in controls and 138 pg in dbcAMP-treated cultured cells. Thus, dbcAMP decreased the total amount of TGF-beta present in the conditioned medium, but increased the concentration of free (active) peptide.

From the above results, cAMP derivatives (such as N6,2-O-dibutyryl cAMP (dbcAMP))
1) causes dose-dependent proliferation promotion of human keratinocytes and human dermal fibroblasts. This mitogenic effect was observed at concentrations of 10-5M and 10-6M and was not affected by the presence of collagen stroma. High concentration (1
The growth inhibition effect observed from 0-3M to 10-4M) was
Addition of EGF or PDGF completely or partially abolished it, whereas addition of TGF-beta increased the inhibitory effect.

[0026] Intracellular levels of cAMP are known to depend on adenylate cyclase and cAMP phosphodiesterase. Adenylate cyclase generates cAMP from ATP, and cAMP phosphodiesterase generates cAMP from ATP.
Change AMP to 5-AMP. Adenylate cyclase activity is known to be regulated by protein kinase C. Certain drugs, such as forskolin, increase intracellular cAMP by acting directly on the catalytic subunit of adenylate cyclase. Other drugs such as 3-isobutyl-methylxanthine (IBMX)
Inhibiting cAMP phosphodiesterase increases intracellular cAMP. In contrast, the cAM of the present invention
P derivative (1) is a cAMP analog that can directly penetrate cell membranes. That is, extracellular addition of dbcAMP directly leads to an increase in intracellular cAMP. The cell growth stimulating effect of the cAMP derivative (1) observed in the present invention is surprising since the theory that cAMP is a cell growth inhibitor has been widely supported. Currently, reports have been published that cyclic nucleotides are positive growth regulators in skin microvascular endothelial cells, B lymphocytes, Schwann cells, epithelial-derived cells, and the like.

Pharmacological test 2 (wound healing effect) (1) Materials and methods (a) Test drug ointment base (Macrogol) and dbcAMP ointment (db
macrogol ointment containing 30mg of cAMP per gram)
was used. (b) Experimental animal male Kb1: using Wistar rats (11 weeks old),
The animals were pre-reared for a week before being used. (c) Administration method The drug was applied transdermally to the wound. The maximum amount applied was 200 mg per location. (d) Test method Rats weighing from 420 g to 479 g were used. After measuring the weight, the animals were anesthetized with ether, and the hair on their dorsal sides was clipped with electric clippers. Rats with no skin abnormalities were selected for experiments. These rats were divided into groups (approximately 15 rats per group) so that the average body weight of all groups was as similar as possible. Under ether anesthesia, the dorsal skin was
A 3.4 cm long midline incision was made using a surgical knife (day 1 was the day the wound was created). The center of the wound, 1 cm cranially from the center, and 1 cm caudal from the center were sutured with sutures, and the sutures were removed 4 days later. The tensile strength of each wound was measured by the method described below on the 5th and 7th day after wound formation. A piece of skin (size: 2 cm width, 3 cm length) including the central part (2 cm) of the wound was excised, and a part 0.5 cm from the edge was fixed. A tensile strength meter (TK-25) designed to measure the tensile strength of wounds for wound treatment.
1, manufactured by Unicom). Once a day from the day of wound formation, administer the test drug for 200 m per wound.
g was applied. The first day was applied immediately after wound formation. 4
On the second day, the drug was applied immediately after the stitches were removed. (e) Statistical analysis All values were expressed as mean ± standard error (SD). The statistical significance of differences was assessed by comparing the control group with other treated groups. p value is 0.0
A value of less than 5 (p<0.05) was considered statistically significant. Analysis of variance was performed using the F test. If the variances were equal, a Student's t-test was performed. In case of unequal variances, Aspin-Welch or Cochran-Cox tests were performed.

(2) Results The results obtained are shown in Tables 1 and 2. The average tensile strength of the wounds in the control group was 11 on the fifth day after wound formation.
1.1 g, and 552.0 g on the 7th day. The average tensile strength of the wounds in the ointment-based group was 1 on day 5.
40.5g and 552.8g on day 7, an increase of 26.5% and 0.1% compared to the values in the control group, respectively.
% increase. The difference in values on day 5 was statistically significant (p<0.05). The average tensile strength of the wound in the dbcAMP ointment group was 196.1 at day 5.
g, and 600.0 g on day 7, which were increases of 76.5% and 8.7%, respectively, compared to the values in the control group. The values on day 5 were significantly higher than those in the control group and ointment base group (p<0.
01). From the above results, the cAMP derivative of the present invention (1
) has the effect of increasing granulation tissue in a wound and promotes wound healing.

[0029]

[Table 1]

[0030]

[Table 2]

Formulation Example 1 (Solution) A solution is prepared by dissolving dbcAMP in 10 mg of physiological saline.

Formulation Example 2 (Ointment) An ointment is prepared by mixing 50 g of Macrogol 4000, 50 g of Macrogol 400, and 3 g of dbcAMP.

[0033]

Effect of the invention: The drug of the present invention has an excellent stimulating effect on the proliferation of keratinocytes and skin fibroblasts, and can cure skin injuries such as surgical wounds, burns, injury wounds, cold injuries, mucosal diseases, and hemorrhoids. promote.

[Brief explanation of the drawing]

[Figure 1] dbcA on human keratinocyte proliferation
The effect of MP is shown by cell number.

FIG. 2 shows the effect of dbcAMP on DNA synthesis in cultured human keratinocytes based on the amount of 3H-thymidine incorporation.

FIG. 3 shows the effect of dbcAMP on DNA synthesis of cultured human skin fibroblasts by the amount of 3H-thymidine incorporation.

FIG. 4 shows the effect of dbcAMP on DNA synthesis of fibroblasts cultured in collagen by the amount of 3H-thymidine incorporation.

FIG. 5: dbcAM on fibroblast DNA synthesis
P and epidermal growth factor (EGF) or platelet-derived growth factor (
The effect of the combination with PDGF) is shown.

FIG. 6: dbcAM on fibroblast DNA synthesis
Figure 2 shows the effect of the combination of P and TGF-beta.

Claims (3)

[Claims] Claim 1: The following general formula (1) [In the formula, R1 represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or an aliphatic acyl group having 2 to 7 carbon atoms, and R2 is hydrogen] atom, mercapto group, alkylthio group having 1 to 6 carbon atoms, benzylthio group, amino group, hydroxyl group, chlorine atom or bromine atom, R3 represents a hydrogen atom or an aliphatic acyl group having 2 to 7 carbon atoms, and X represents Indicates a hydrogen atom or an alkali metal atom. However, R1, R2 and R3 do not become hydrogen atoms at the same time. ] Adenosine-3 expressed as
A growth stimulant for keratinocytes and skin fibroblasts containing a ',5'-cyclic phosphoric acid derivative as an active ingredient. 2. A growth stimulant according to claim 1 in external form. [Claim 3] Adenosine-3',5 according to claim 1
A growth stimulant for keratinocytes and skin fibroblasts, comprising a '-cyclic phosphate derivative and epidermal growth factor or platelet-derived growth factor.